1. Field of the Invention
The invention relates generally to immobilization of enzymes in a carrageenan matrix. In particular aspects, carrageenan immobilized enzymes effectively catalyze reactions in organic solvents. Typical reactions include stereoselective transesterification and hydrolysis reactions.
The following is a table of abbreviations used.
TABLE 1 ______________________________________ ee enantiomeric excess HPLC High performance liquid chromatography PLE Pig liver esterase MOPSO 3-[N-morpholino]-2- hydroxypropanesulfonic acid THF tetrahydrofuran ETOAc ethyl acetate MeOH methyl alcohol KOAc potassium acetate DMF dimethyl formamide HCl hydrochloric acid ______________________________________
2. Description of Related Art
Historically enzymes wee thought to function only in aqueous media; however it was shown by Klibanov (1985) and subsequently by others (Inaday et al, 1986; Luisi, 1985) that enzymes can and do function in organic solvents. This discovery has stimulated interest in developing enzyme systems that can be used to efficiently perform organic reactions, particularly reactions that are stereoselective and that without enzymes are difficult to perform. Enzymes have been used in aqueous systems to perform chemical reactions, for example, transesterifications and isomerizations. Some reactions have been achieved in organic solvents. There is increasing recognition of the value of enzymatic catalysis in organic solvents, especially since selectivity is often increased compared to the reaction in aqueous media. As noted in a review by Wong (1989), enzyme catalyzed dehydrations, transesterifications, aminolyses, and oxidoreductions in organic solvents are becoming more common.
However, there are drawbacks to using enzymes in organic solvents. In some cases a relatively large amount of enzyme is required, especially when powdered preparations are used. Vigorous stirring may also be required in order to assure adequate contact with the dissolved substrate. Enzyme preparations from powdered or freeze dried enzymes may suffer from lower activity and therefore function inefficiently. In some instances, enzymes may be inactivated in an organic solvent to the extent that catalyzed reactions, if they occur at all, are too slow to be useful.
Enzymes immobilized within various matrices have been widely used to catalyze reactions in aqueous systems (Schneider et al, 1985; Chibata et al, 1984); however, few have been developed for use in nonaqueous systems. In one of few examples, selected enzymes immobilized by gel entrapment in polyacrylamide cross linked with N,N'-methylenebisacrylamide yielded reverse micelles useful in water immiscible solvents such as isooctane (Fadnavis and Luisi, 1989). There has been some success in enhancing enzyme stability in water miscible organic solvents. In one method, a soluble enzyme was coated with polymeric glutaraldehyde and the layer then crosslinked with a second layer of polyacrylamide derivatives to create a synthetic cage around the enzyme (Tor et al, 1989).
A few processes have been developed using immobilized enzymes in organic solvents, often in membrane reactors. Essentially, an enzyme is imbedded in a matrix which is then exposed to a substrate dissolved in an appropriate solvent. A multiphase membrane reactor has been described (Matson, 1989) in which organic or aqueous soluble substrates may be transformed as they contact the membrane immobilized enzyme.
Despite the development of membrane reactors, there are drawbacks to using this method. The reactor design is usually elaborate and costly. A frequently encountered problem is membrane plugging which then interferes with mass transport.
There is, therefore, a need to develop methods of enzyme immobilization providing stable, efficient, easily handled enzymes, particularly immobilized forms which stabilize an enzyme in organic solvents. Such immobilized enzymes could catalyze numerous organic reactions capable of providing potentially large quantities of optically enriched materials useful either as intermediates or as products for industries producing pharmaceuticals, pesticides, and herbicides.